EP3899534A1 - Procédé de conservation de cellules urinaires - Google Patents

Procédé de conservation de cellules urinaires

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Publication number
EP3899534A1
EP3899534A1 EP19821138.5A EP19821138A EP3899534A1 EP 3899534 A1 EP3899534 A1 EP 3899534A1 EP 19821138 A EP19821138 A EP 19821138A EP 3899534 A1 EP3899534 A1 EP 3899534A1
Authority
EP
European Patent Office
Prior art keywords
cells
urine
urine sample
buffer substance
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP19821138.5A
Other languages
German (de)
English (en)
Other versions
EP3899534C0 (fr
EP3899534B1 (fr
Inventor
Philipp Enghard
Paul Freund
Diana METZKE
Christopher SKOPNIK
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Charite Universitaetsmedizin Berlin
Original Assignee
Charite Universitaetsmedizin Berlin
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Publication date
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Publication of EP3899534A1 publication Critical patent/EP3899534A1/fr
Application granted granted Critical
Publication of EP3899534C0 publication Critical patent/EP3899534C0/fr
Publication of EP3899534B1 publication Critical patent/EP3899534B1/fr
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Anticipated expiration legal-status Critical

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1404—Handling flow, e.g. hydrodynamic focusing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders

Definitions

  • the present invention relates to a method for preserving urine cells.
  • Flow cytometry fluorescence-activated cell sorting, FACS
  • FACS fluorescence-activated cell sorting
  • the present invention aims to provide means and methods to preserve cells in a urine sample in such a way that they are stable for more than 4 to 6 hours prior to analysis but are also suitable for an analysis, e.g. by antibody based methods such as FACS.
  • the sample treatment according to the invention would allow the storage and/or transport of a urine sample prior to the analysis.
  • the preservation does not change the cells in such a way that no meaningful staining for flow cytometric analysis is possible.
  • This objective is attained by using a buffer substance to adjust and maintain the pH between pH 6 and pH 8 and by adding a formaldehyde releasing compound to the urine sample.
  • Cells that are present in the urine may be detected or analyzed in a subsequent analysis step.
  • a suitable method for such detection or analysis is FACS.
  • a first aspect of the invention relates to a method for analysing urinary cells.
  • the method comprises the steps of:
  • a buffer substance suitable to create and/or maintain a pH value in the range of 6 to 8, particularly approx. pH 7, within said urine sample, and
  • Another aspect of the invention relates to a method for diagnosing a medical condition.
  • the method comprises analysing urine cells within a urine sample obtained from a patient with a method according to the first aspect of the invention.
  • Yet another aspect of the invention relates to a container suitable for preserving urinary cells in a urine sample comprising a formaldehyde releasing compound, and a buffer substance able to maintain at a pH in the range of 6 to 8, particularly approx. 7, in a urine sample.
  • Reference to“about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to“about X” includes description of“X.”
  • buffer substance in the context of the present specification is used in its meaning known in the art; it particularly refers to a weak acid or base and it conjugate base or acid.
  • a suitable buffer substance to create and/or maintain a pH value in the range of 6 to 8 is preferable characterized by at least one PK A value in the range of 6 to 8.
  • formaldehyde releasing compound in the context of the present specification particularly refers to a compound that releases formaldehyde, particularly slowly but not spontaneously, for example in the presence of water or a suitable aqueous buffer solution.
  • Suitable formaldehyde releasing compounds are characterized by a formaldehyde release of up to 20 mg/L, particularly 1 mg/L to 15 mg/L, more particularly 5 mg/L to 15 mg/L, at one hour after dissolving 0.1 % (w/v) of the formaldehyde releasing compound in an aqueous solution (pH 7) at 25 °C.
  • Formaldehyde releasing compounds are also commonly known as formaldehyde releasers.
  • the term “formaldehyde releasing compound” excludes formaldehyde and paraformaldehyde.
  • urinary cells in the context of the present specification refers to cells that are found in the urine. The term also encompasses cells that not naturally present in urine, for example cancer cells, etc.
  • the urinary cells are mammalian cells, particularly human cells.
  • a first aspect of the invention relates to a method for preserving urinary cells.
  • the method comprises the step of:
  • the urine sample is contacted simultaneously with the buffer substance and the formaldehyde releasing compound or the urine sample is first contacted with the buffer substance and then the formaldehyde releasing compound is added.
  • the urine sample is contacted simultaneously with the buffer substance and the formaldehyde releasing compound.
  • the urine sample is first contacted with the buffer substance and then the formaldehyde releasing compound is added.
  • the method consists of the step of contacting a urine sample obtained from a patient with a buffer substance suitable to create and/or maintain a pH value in the range of pH 6 to pH 8 within the urine sample, and a formaldehyde releasing compound.
  • the buffer substance is suitable to create and/or maintain a pH value in the range of 6.9 to 7.4 within the urine sample.
  • the said urine sample is contacted with the buffer substance and the formaldehyde releasing compound not later than 6h, 5h, 4h, 3h, 2h, or 1 h after sampling, particularly immediately after sampling.
  • the buffer substance is in such a concentration when contacted with the urine sample that within the urine sample the desired pH value, e.g. 7.2 to 7.4, forms without need of further adjustment, e.g. by additionally adding an acid or a base.
  • the formaldehyde releasing compound is selected from imidazolium urea (CAS No 39236-46-9), hexamethylenetetramine chloroallyl chloride (CAS No 4080-31- 3), diazolidinyl urea (CAS No 78491-02-8), 1 ,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine- 2,4-dione (DMDM hydantoin, CAS No 6440-58-01 ), 1-(hydroxymethyl)-5,5-dimethyl-2,4- imidazolidindione (MDM hydantoin) or hexamethylenetetramine (urotropin, CAS No 100-97- 0).
  • the formaldehyde releasing compound is not paraformaldehyde.
  • the buffer substance is selected from 3-(N- morpholino)propanesulfonic acid (MOPS), phosphate buffered saline (PBS), sodium hydrogencarbonate, tris(hydroxymethyl)aminomethane (TRIS) and 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES).
  • MOPS 3-(N- morpholino)propanesulfonic acid
  • PBS phosphate buffered saline
  • TMS tris(hydroxymethyl)aminomethane
  • HEPES 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid
  • the buffer substance is selected from 3-(N- morpholino)propanesulfonic acid (MOPS), phosphate buffered saline (PBS), and 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).
  • MOPS 3-(N- morpholino)propanesulfonic acid
  • PBS phosphate buffered saline
  • HEPES 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid
  • the buffer substance is provided in a buffer solution.
  • the concentration of the buffer substance in the buffer solution is between 0.2 mol/l and 5 mol/l.
  • the concentration of the buffer substance in the buffer solution is between 0.5 mol/l and 2 mol/l.
  • the concentration of the buffer substance in the buffer solution is 1 mol/l.
  • the urine sample is further contacted with a chelating compound selected from 2,2',2",2"'-(ethane-1 ,2-diyldinitrilo)tetraacetic acid (EDTA), ethylene glycol- bis(2-aminoethylether)-/V,/V,/V',/V'-tetraacetic acid (EGTA) and 2,2',2"-nitrilotriacetic acid (NTA).
  • EDTA 2,2',2",2"'-(ethane-1 ,2-diyldinitrilo)tetraacetic acid
  • EGTA ethylene glycol- bis(2-aminoethylether)-/V,/V,/V',/V'-tetraacetic acid
  • NTA 2,2',2"-nitrilotriacetic acid
  • the urine sample is further contacted with EDTA.
  • the buffer substance may be added to the urine sample before the formaldehyde releasing compound is added.
  • the urine sample is contacted first with the buffer substance and then with the formaldehyde releasing compound.
  • the final concentration of the formaldehyde releasing compound in the preserved sample directly after the contacting step is between 0.5 % (w/v) and 5 % (w/v). The preservation does not change the cells in such a way that no meaningful staining for flow cytometric analysis is possible.
  • the final concentration of the formaldehyde releasing compound is between 0.5 % (w/v) and 5 % (w/v).
  • the final concentration of the formaldehyde releasing compound is between 1 % (w/v) and 3 % (w/v).
  • the final concentration of the formaldehyde releasing compound is 2 % (w/v).
  • the final concentration of the buffer substance in the preserved sample directly after the contacting step is between 5 % (w/v) and 10 % (w/v).
  • the final concentration of the buffer substance is between 5 % (w/v) and 10 % (w/v).
  • the final concentration of the buffer substance is 7 % (w/v).
  • the final concentration of the buffer substance is between 5 % (w/v) and 10 % (w/v), particularly 7 % (w/v), and the final concentration of the formaldehyde releasing compound is between 0.5 % (w/v) and 5 % (w/v), particularly between 1 % (w/v) and 3 % (w/v).
  • the preserved sample may be stored at 4 °C up to one month prior to analysing the cells. It is also possible to freeze the preserved sample.
  • the preserved sample is stored prior to analysing the urinary cells.
  • the preserved sample is stored prior to analysing the urinary cells at a temperature ⁇ 10 °C, particularly ⁇ 4 °C.
  • the preserved sample is stored prior to analysing the urinary cells at a temperature between -80 °C and 10 °C, particularly -80 °C and 4 °C.
  • the preserved sample is stored prior to analysing the urinary cells at a temperature between -20 °C and 10 °C, particularly -20 °C and 4 °C.
  • a second aspect of the invention relates to a method for analysing urinary cells.
  • the method comprises the steps of:
  • analysing comprises a cytometric analysis of the urinary cell in the preserved urine sample, e.g. measurement of cell size, cell count cell morphology (shape and structure), cell cycle phase, DNA content, cell surface proteins, etc.
  • cytometric analysis e.g. measurement of cell size, cell count cell morphology (shape and structure), cell cycle phase, DNA content, cell surface proteins, etc.
  • the cyctometric analysis is or comprises a flow cytometric analysis, particularly fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • a third aspect of the invention relates to a method for diagnosing a medical condition.
  • the method comprises analysing urinary cells in a urine sample obtained from a patient with a method according to the second aspect of the invention or any embodiment thereof.
  • the medical condition is selected from Lupus nephritis, acute Kidney Injury, rejection after kidney transplantation, ANCA vasculitis, diabetic nephropathy, IgA nephropathy, nephrolithiasis (renal stones), and bladder cancer.
  • the medical condition is Lupus nephritis and the presence and/or quantity of a surface marker selected from CD3, CD4, CD8, CD45RA, CD45RO, CCR7, CD14, CD36 .is determined on the urinary cells within the preserved urine sample.
  • the medical condition is renal transplant rejection and the presence and/or quantity of a surface marker selected from CD3, CD4, CD8, CD45RA, CD45RO, CCR7, CD14, CD69 CD36, Podocalyxin, Cytokeratin, CD10, CD13, EPCAM, CD227 is determined on the urinary cells within the preserved urine sample.
  • a surface marker selected from CD3, CD4, CD8, CD45RA, CD45RO, CCR7, CD14, CD69 CD36, Podocalyxin, Cytokeratin, CD10, CD13, EPCAM, CD227 is determined on the urinary cells within the preserved urine sample.
  • the medical condition is acute kidney injury and the presence and/or quantity of a surface marker selected from CD3, CD4, CD8, CD45RA, CD45RO, CCR7, CD14, CD36, Podocalyxin, Cytokeratin, Podocin (PDCN, SRN1 ) nephrin (NPHN, NPHS1 ), Wilms Tumour Protein (WT1 ), CD10, CD13, EPCAM, CD227 is determined on the urinary cells within the preserved urine sample.
  • a surface marker selected from CD3, CD4, CD8, CD45RA, CD45RO, CCR7, CD14, CD36, Podocalyxin, Cytokeratin, Podocin (PDCN, SRN1 ) nephrin (NPHN, NPHS1 ), Wilms Tumour Protein (WT1 ), CD10, CD13, EPCAM, CD227 is determined on the urinary cells within the preserved urine sample.
  • the medical condition is ANCA associated glomerulonephritis and the presence and/or quantity of a surface marker selected from CD3, CD4, CD8, CD45RA, CD45RO, CCR7, CD14, CD36, Podocalyxin, Cytokeratin, CD10, CD13, EPCAM, CD227 is determined on the urinary cells within the preserved urine sample.
  • a surface marker selected from CD3, CD4, CD8, CD45RA, CD45RO, CCR7, CD14, CD36, Podocalyxin, Cytokeratin, CD10, CD13, EPCAM, CD227 is determined on the urinary cells within the preserved urine sample.
  • a fourth aspect of the invention relates to a container suitable for preserving urinary cells in a urine sample.
  • the container comprises
  • a formaldehyde releasing compound a formaldehyde releasing compound, and a buffer substance able to create and/or maintain at a pH in the range of 6 to 8 in a urine sample.
  • the container comprises an inner volume, wherein a composition is present in said inner volume and wherein the composition comprises, particularly consists of, a formaldehyde releasing compound selected from imidazolium urea,
  • hexamethylenetetramine chloroallyl chloride diazolidinyl urea, 1 ,3-bis(hydroxymethyl)-5,5- dimethylimidazolidine-2,4-dione, 1 -(hydroxymethyl)-5,5-dimethyl-2,4-imidazolidindione or hexamethylenetetramine, and
  • a buffer substance able to maintain at a pH in the range of pH 6 to pH 8, particularly approx. pH 7, in a urine sample.
  • the container is configured to receive and hold a fluid, particularly a urine sample.
  • the container is a tube and comprises a sealable opening, through which the urine sample may be transferred into the container.
  • the container may be a urine cup.
  • the patient or medical staff can add the urine specimen or sample immediately to the container and store it directly in the fridge after inverting it several times.
  • the formaldehyde releasing compound and / or the buffer substance are present in the container in solid form, e.g. in form of a powder, tablets or a granulate.
  • the formaldehyde releasing compound is selected from imidazolium urea, hexamethylenetetramine chloroallyl chloride, diazolidinyl urea, 1 ,3-bis(hydroxymethyl)- 5,5-dimethylimidazolidine-2,4-dione., 1-(hydroxymethyl)-5,5-dimethyl-2,4-imidazolidindione or hexamethylenetetramine.
  • the formaldehyde releasing compound is not paraformaldehyde.
  • the buffer substance is selected from 3 -(/V- morpholino)propanesulfonic acid, phosphate buffered saline, sodium hydrogencarbonate, tris(hydroxymethyl)aminomethane and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
  • the buffer substance is selected from 3 -(/V- morpholino)propanesulfonic acid, phosphate buffered saline, sodium hydrogencarbonate, and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
  • the ratio of the amount of formaldehyde releaser to the amount of buffer substance is between 1 :10 and 1 :2, particularly between 1 :5 and 2:5. In certain embodiments, the ratio of the amount of formaldehyde releaser to the amount of buffer substance is 2:7.
  • Embodiment 1 A method for preserving urinary cells, the method comprising the step of:
  • Embodiment 2 The method according to embodiment 1 , wherein said urine sample is contacted with said buffer substance and said formaldehyde releasing compound not later than 6h, 5h, 4h, 3h, 2h or 1 h after sampling, particularly immediately after sampling.
  • Embodiment 3 The method according to embodiment 1 or 2, wherein said formaldehyde releasing compound is selected from imidazolium urea, hexamethylenetetramine chloroallyl chloride, diazolidinyl urea, 1 ,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione., 1- (hydroxymethyl)-5,5-dimethyl-2,4-imidazolidindione or hexamethylenetetramine.
  • said formaldehyde releasing compound is selected from imidazolium urea, hexamethylenetetramine chloroallyl chloride, diazolidinyl urea, 1 ,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione., 1- (hydroxymethyl)-5,5-dimethyl-2,4-imidazolidindione or hexamethylenetetramine.
  • Embodiment 4 The method according to any one of the preceding embodiments, wherein said suitable buffer substance is selected from 3-(/V-morpholino)propanesulfonic acid, phosphate buffered saline sodium hydrogencarbonate, tris(hydroxymethyl)aminomethane and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
  • said suitable buffer substance is selected from 3-(/V-morpholino)propanesulfonic acid, phosphate buffered saline sodium hydrogencarbonate, tris(hydroxymethyl)aminomethane and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.
  • Embodiment 5 A method for analysing urinary cells, comprising
  • preserving said urine sample with a method according to any one of embodiments 1 to 4, thereby yield a preserved urine sample, and analysing urinary cells within said preserved urine sample.
  • Embodiment 6 The method according to embodiment 5, wherein said analysing comprises a cytometric analysis of said urinary cells with said preserved urine sample.
  • Embodiment 7 The method according to embodiment 6, wherein said cytometric analysis comprises a flowcytometric analysis, particularly FACS.
  • Embodiment 8 A method for diagnosing a medical condition, the method comprising analysing urine cells within a urine sample obtained from a patient with a method according to any one of embodiments 5 to 7.
  • Embodiment 9 The method according to embodiment 8, wherein said medical condition is selected from Lupus nephritis, acute kidney injury, rejection after kidney transplantation, ANCA vasculitis, diabetic nephropathy, IgA nephropathy, nephrolithiasis, and bladder cancer.
  • Embodiment 10 A container suitable for preserving urinary cells in a urine sample
  • a buffer substance able to maintain at a pH in the range of 6 to 8, particularly approx. 7, in a urine sample.
  • Embodiment 1 1 The container according to embodiment 10, wherein said formaldehyde releasing compound is selected from imidazolium urea, hexamethylenetetramine chloroallyl chloride, diazolidinyl urea, 1 ,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione., 1- (hydroxymethyl)-5,5-dimethyl-2,4-imidazolidindione or hexamethylenetetramine.
  • said formaldehyde releasing compound is selected from imidazolium urea, hexamethylenetetramine chloroallyl chloride, diazolidinyl urea, 1 ,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione., 1- (hydroxymethyl)-5,5-dimethyl-2,4-imidazolidindione or hexamethylenetetramine.
  • Embodiment 12 The container according to embodiment 10 or 11 , wherein said buffer substance is selected from 3-(/V-morpholino)propanesulfonic acid, phosphate buffered saline, sodium hydrogencarbonate, tris(hydroxymethyl)aminomethane and 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid.
  • said buffer substance is selected from 3-(/V-morpholino)propanesulfonic acid, phosphate buffered saline, sodium hydrogencarbonate, tris(hydroxymethyl)aminomethane and 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid.
  • Fig. 1 shows the relation between pH values and precipitate formation in urine. Especially specimen with low pH values tend to form precipitation.
  • A Example of precipitation of patient’s urine with added formaldehyde after 22 hours (+/- 2 hours).
  • B Dipstick results and relation to precipitation of urine with added formaldehyde after 22 hours (+/- 2 hours)
  • Fig. 2 shows representative dot plots of peripheral blood mononuclear cells (PBMCs) in urine show reduced staining quality after incubation with MOPS buffer and formaldehyde for 22 hours (+/- 2 hours).
  • Gating strategy for T Cells lymphocyte scatter-gate. Selection of CD3+ T Cells. Discrimination CD4+ T Cells vs. CD8+ T Cells.
  • Gating strategy for macrophages monocyte scatter-gate. Selection of CD14+ CD36+ macrophages.
  • A Fresh PBMCs, fully stained.
  • B PBMCs incubated in urine with MOPS buffer and 1 % formaldehyde for 22 hours (+/- 2 hours)
  • Fig. 3 shows representative dot plots of urinary leukocytes. Staining quality
  • CD4+ T cells vs. CD8+ T Cells. Gating strategy for macrophages: exclusion of debris (low FSC and SSC) and large events (very high FSC and SSC). Selection of CD14+ CD36+ macrophages.
  • A Fresh patient specimen ( ⁇ 6 hours after voiding), unstained.
  • B Fresh patient specimen ( ⁇ 6 hours after voiding), stained.
  • C conserveed patient specimen after 22 hours (+/- 2 hours) at 4 °C, stained.
  • Fig. 4 shows representative dot plots of urinary epithelial cells. Staining quality
  • TECs tubular epithelial cells
  • Fig. 5 shows the staining quality and percentages of populations of urinary leukocytes from conserved sample remain stable for up to 6 days, whereas leukocyte populations in non-fixated urine specimen disappear after 6 days (see Fig. 4 D).
  • Gating strategy for T cells Exclusion of debris (low FSC and SSC) and large events (very high FSC and SSC). Selection of CD3+ T Cells. Discrimination CD4+ T Cells vs. CD8+ T cells.
  • Gating strategy for macrophages Exclusion of debris (low FSC and SSC) and large events (very high FSC and SSC). Selection of CD14+ CD36+ macrophages. Representative FACS plots shown.
  • Fig. 6 shows the staining quality and percentages of populations of urinary epithelial cells from conserved sample remain stable for up to 6 days, whereas cell populations in non-fixated urine specimen almost disappear after 6 days (see Fig. 5 D).
  • A conserveed patient specimen after 22 hours (+/- 2 hours) at 4 °C.
  • B conserveed patient specimen after 3 days at 4 °C.
  • C conserveed patient specimen after 6 days at 4 °C.
  • D Patient specimen without conservation after 6 days at 4 °C.
  • Fig. 7 shows the total cell counts of analyzed urinary leukocyte and epithelial cell
  • Fig. 8 shows the preservation of cells in urine.
  • A Representative flow cytometry plots of staining on fresh cells and after 20h preservation. No qualitative deterioration of staining is observed.
  • B Comparison of cell numbers in urine in the freshly measured sample and after 24h preservation, each point represents one sample. The gray line shows absolutely identical cell numbers, the dashed line shows the regression analysis of the actually measured values. There is a high degree of agreement, i.e. an almost constant cell count despite preservation.
  • Fig. 9 shows the kinetics of the fixation of immune cells using IU. Displayed is the
  • Fig. 10 shows CD4 + T cells as biomarkers for active lupus nephritis (LN).
  • A Correlation of CD4 + T cell count per 100 ml urine with disease activity (measured as SLEDAI). Each open square represents one SLE patient without renal involvement, filled circles represent one SLE patient with known renal involvement.
  • B Comparison of the amount of CD4 + T cells in the urine with a simultaneous kidney biopsy. All patients with inflammatory LN (proliferative LN) show increased levels of T cells in urine.
  • C ROC curve for the detection of proliferative/inflammatory LN in SLE patients using the amount of CD4 + T cells in urine (sensitivity of 100% and specificity of 98.0%).
  • D Correlation of CD4 + T cell count per 100 ml urine with disease activity (measured as SLEDAI). Each open square represents one SLE patient without renal involvement, filled circles represent one SLE patient with known renal involvement.
  • B Comparison of the amount of CD4 + T cells in the urine with a simultaneous kidney biopsy
  • Fig. 11 shows different cell populations in urine as markers for acute kidney transplant rejection.
  • A Representative flow cytometry plots for the detection of T-cells (CD3, CD4, CD8), macrophages (CD45, CD36, CD14), podocalyxin-positive cells (PDX, as surrogate for podocytes) and renal tubule epithelial cells (TEC; cytokeratin,
  • CD10 EPCAM
  • B Amount of T cells in relation to TEC and PDX + cells in patients with acute cellular rejection (ACR), humoral rejection (HRX), patients with graft degeneration without rejection (No Rx) and patients with stable graft function (Contr.).
  • C ROC curves show a good diagnostic significance of the combination of T cells and TECs or PDX + cells in urine (AUC 0.9).
  • Black line comparison of patients with rejection with the No RX group; grey line: data including contrast patients with stable graft function.
  • Example 1 Preserving and analyzing urine cells
  • Fixation of cells and tissues with agents such as formaldehyde is considered routine practise in laboratories.
  • the basis of this fixation is cross-linking of proteins at certain amino acids.
  • MOPS standard cell culture buffer
  • formaldehyde releasers such as, but not limited to, imidazolidinyl urea (IU) instead of direct addition of formaldehyde.
  • the chemical group of formaldehyde releasers is used for example as a preservative in cosmetic products and is characterized by a slow release of formaldehyde- groups from a more complex molecule.
  • staining quality was only marginally impaired in comparison to fresh urine samples (Figs. 3 - 6).
  • the inventors were able to detect similar counts of urinary cells after 22 hours (+/- 2 hours) compared to fresh samples ( Figure 7A). Furthermore, detected similar numbers of urinary cells were detected even after 3 and 6 days compared to samples analysed after 1 day ( Figure 7B and 7C).
  • the sample can be stored at 4 °C for 22 hours, 3 days, 6 days or potentially even longer for later sample preparation, i.e. antibody-based staining of cells, and flow cytometric analysis.
  • the method of the invention allows delayed sample preparation and analysis of human urinary cells through an easy conservation procedure. Both components (IU and MOPS buffer) are simply added to the urine specimen. After inverting the sample several times it can be stored in the fridge (4 °C) for 22 hours, 3 days, 6 days or potentially even longer. This enables centralized and standardized analysis facilitating multicentre studies as well as future diagnostic use in more peripheral clinics and outpatient care. Another important advantage of the method of the invention is increased safety when working with human samples, as cells will be dead after fixation. Furthermore, the conservation procedure leads to fixation of the cells, thus enabling to start directly with permeabilization for intracellular staining after a centrifugation step.
  • the inventors have analyzed 15 samples for T Cell counts (which stain positive for CD3 and CD4 or CD3 and CD8) and Macrophages (positive for CD14 and CD36) and 9 samples for counts of kidney-derived tubular epithelial cells (positive for cytokeratin and CD326 (EpCAM) or cytokeratin and CD10 and CD13), respectively, after 22 hours (+/- 2 hours). For time points after 3 days and 6 days, the inventors have acquired data from 10 samples for both T cells and tubular epithelial cells, respectively. Every urine specimen was split equally allowing to compare cell counts of the conservation method of the invention after a certain time to a reference sample.
  • Specimen were acquired from patients with diverse renal pathologies, including: Acute Kidney Injury (AKI), Diabetic Nephropathy, Acute Rejection of a Kidney Transplant,
  • AKI Acute Kidney Injury
  • Diabetic Nephropathy Diabetic Nephropathy
  • Acute Rejection of a Kidney Transplant Acute Rejection of a Kidney Transplant
  • the method according to the invention can be used for any renal or urologic disease or any disease that leads to altered cell composition in the urine.
  • IU imidazolidinyl urea
  • Example 2 Establishment of a sample preservation system
  • the difficulty here was not only to preserve the cells, but also not to change the cells in such a way that no meaningful staining for flow cytometry is possible after preservation.
  • the preservation system must be as simple and uncomplicated as possible in order to be accepted by the users (medical personnel in routine operation).
  • the inventors succeeded in preserving the cells for at least six days. All that is required is to add a urine sample to a container containing a buffer and then add a formaldehyde releasing compound (in powder or tablet form).
  • the container is equipped with the buffer compound and formaldehyde releasing compound in a way that enables stable shelf life for both, and the urine sample is added, optionally after addition of water, upon opening of the container from its packaging.
  • the sample can then be stored and/or transported. With this preservation, staining and flow cytometry of the cells can be performed without compromising the quality of the analysis.
  • the system is user-friendly and does not require a complicated or time-consuming protocol.
  • WO 2014/029791 A1 shows a flow cytometric analysis of cells that have been treated with different variants of a fixation protocol. The following reagents were used to preserve the cells and each solution was A to E was diluted 1 :50 with the cell sample:
  • the mentioned variants for cell preservation were compared with the protocol according to the invention, i.e. the solutions A to E were each diluted 1 :50 with urine.
  • Aurintricarboxylic acid rapidly precipitates when added to urine, rendering it unusable for the purpose of the protocol of the present invention.
  • EDTA alone (0.2 % final concentration) precipitates in urine.
  • IU fixation was performed with different incubation times: 5min, 30min, 1 h, 5h, 1 day.
  • LN Lupus nephritis
  • SLE systemic lupus erythematosus
  • MMF mycophenolate mofetil
  • EM T cells memory/effector T cells
  • lupus nephritis can be i) diagnosed with T-cells in urine, ii) the response to treatment monitored and iii) the prognosis estimated.
  • Kidney transplantation is probably the best therapy for terminal renal failure.
  • acute rejection of the transplant is a constant concern, requiring permanent immunosuppressive use and regular monitoring of graft function.
  • there are no reliable non-invasive markers for acute rejection of the transplant which is why a kidney biopsy with

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Abstract

La présente invention concerne un procédé de conservation de cellules urinaires, le procédé comprenant l'étape consistant à mettre en contact un échantillon d'urine obtenu à partir d'un patient avec une substance tampon appropriée pour créer et/ou maintenir une valeur de pH dans la plage de 6 à 8, en particulier environ 7, à l'intérieur dudit échantillon d'urine, et un composé libérant du formaldéhyde.
EP19821138.5A 2018-12-19 2019-12-19 Procédé pour préserver des cellules urinaires Active EP3899534B1 (fr)

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US5459073A (en) * 1991-05-08 1995-10-17 Streck Laboratories, Inc. Method and composition for preserving antigens and process for utilizing cytological material produced by same
US6794152B2 (en) * 2000-12-22 2004-09-21 Streck Laboratories Inc. Flow cytometry reagent and system
WO2014029791A1 (fr) * 2012-08-21 2014-02-27 Qiagen Gmbh Procédé pour isoler des acides nucléiques à partir d'un échantillon stabilisé libérant du formaldéhyde
US11168351B2 (en) * 2015-03-05 2021-11-09 Streck, Inc. Stabilization of nucleic acids in urine
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